Accumulating evidence suggests that CD4+CD25+ regulatory T cells (Tregs) are an essential component of immune regulation, and loss of Treg functions may be one of the underlying causes of autoimmunity. However, how Tregs suppress immune responses in vivo remains unclear. This is likely due to the low frequency of Tregs and the difficulty in analyzing in vivo responses. We have recently developed a robust system to restore Tregs and control autoimmune diabetes in non-obese diabetic mice by transferring in vitro expanded islet antigen-specific Tregs purified from the BDC2.5 TCR transgenic mice. We hypothesize that Tregs both control immune homeostasis in steady state and respond to inflammatory stimuli to suppress immune responses at local tissue sites. New imaging technologies offer the most direct and unmanipulated ways to analyze in vivo cellular activities. However the field is facing the challenges of moving into clinically relevant disease models and establishing causal relationships between various imaging observations with functional outcomes. We will directly analyze both the steady-state and inflammatory responses of Tregs in vivo in our model system using novel in vivo imaging technologies. Specifically, we will use bioluminescence to monitor the lymph node homing and tissue trafficking of luciferase-tagged diabetogenic CD4 and CDS T cells in the presence and absence of co-transferred BDC Tregs. We will analyze the priming and effector function development of the diabetogenic T cells using two-photon laser scanning microscopy and determine the effect of BDC Tregs on these activities in both LN and islet tissues. We will directly visualize Treg interaction with antigen presenting cells and effector T cells in vivo with two-photon microscopy to determine the direct cellular target of the Tregs. Finally, we will examine the roles of immunosuppressive molecules IL-10, TGF- beta, and CTLA-4 using this novel technology and determine the effect of these molecules on the homing, trafficking, priming, and effector function development of the diabetogenic T cells. We will combine the imaging analysis with conventional cellular immunological approaches, multicolor immunofluorescence and immunohistochemistry techniques to pinpoint the time and location of Treg activation and function. The results of these studies will provide new insights into our understanding of autoimmune diseases by specifically studying the balance between immune regulation and auto-aggression